GSM8084326: midgut-negative1; Cimex lectularius; RNA-Seq
Midgut and RoB tissues (RoB = rest of the body: bodies without heads and midgut tissues) of treatment and control bed bugs were dissected 12 h after an immune challenge, and total RNA was extracted using TRizol reagent (Invitrogen) following the manufacturer's recommendations. The samples were quantified on a Nanodrop 1000 spectrophotometer v. 3.7 (Thermo Fisher Scientific, USA). RNA samples were used to generate complementary DNA (cDNA) for analysis using quantitative real-time polymerase chain reaction (qPCR) and transcriptome assembly. We created a de novo transcriptome assembly from RNA extracted from midgut and RoB tissues of male bed bugs 12 h after ingesting blood containing E. coli or B. subtilis. RNA purification, first and second strand synthesis, adaptor ligation, quantification, validation, and Illumina sequencing were all done at GENEWIZ LLC. (South Plainfield, NJ, USA). The RNA samples were sequenced using a paired-end (PE) configuration, consisting of two 150 base pair (bp) strands. HiSeq Control Software (HCS) was used for image analysis and base calling. Raw sequence data (.bcl files) generated from Illumina HiSeq were converted into fastq files and demultiplexed with bcl2fastq 2.17 software (Illumina Inc, San Diego, CA, USA). For index sequence identification, one mismatch was allowed. After examining the quality of raw data, adapter sequences and nucleotides of poor quality were removed from sequence reads using Trimmomatic v.0.36. The trimmed reads were mapped to the reference genome available on ENSEMBL, using STAR aligner v.2.5.2b which utilizes a splice aligner to detect splice junctions and incorporates them to help align the entire read sequences to generate Binary Alignment Map (BAM) files. In the Subread package version 1.5.2, we used the 'Counts' feature to calculate the unique gene count. Only reads predicted to fall within exons were counted.